Figure 1
Figure 1. Phenotypic isolation of osteoblasts. (A) Strategy for isolation of bone-associated cells. (B) Flow cytometric analysis of single-cell suspensions from collagenase-treated bone (Bone) or BM aspirates (BM). Data are presented as dot plots showing staining for hematopoietic markers (CD45/Ter119) and Opn. The frequency of Opn+CD45−Ter119− cells is shown in the upper left. “-CNTRL” indicates representative background fluorescence seen in the absence of Opn secondary antibody (rat-αFITC) or when an isotype control (rat-αIgG) only is used. This is representative of 10 to 15 independent experiments. (C) QRT-PCR analysis of mRNA isolated from double-sorted Opn+ cells (OPN+) compared with irrelevant bone marrow cells (BM) or Opn− (OPN−) cells. Relative expression (mean ± SD) over a β-actin normalization control is shown for osteocalcin (OC), alkaline phosphatase (ALP), osteoactivin (OA), Runx2 (RX2), and osterix (OS), vascular endothelial growth factor receptor 1 and 2 (VEGFR1, VEGFR2), tyrosine kinase with Ig and EGF homology domains 1 and 2 (TIE1, TIE2), endothelial nitric oxide synthase (eNOS); n = 3 independent experiments for osteolineage markers; n = 2 independent experiments for endothelial markers). Each transcript was measured in triplicate. (D) Flow cytometric analysis of parathyroid hormone receptor-1 (PTHR-1) expression by Opn+CD45−Ter119− cells (gray histogram) or BM cells (black line). Both PTHR1hi and PTHR1lo OPN−CD45−Ter119− cells exhibited equal levels of activity of bone-specific ALP (Figure S2), consistent with an equivalent state of osteolineage differentiation of PTHR1hi and PTHR1lo cells12,42 and probably reflecting known differences in PTHR1 expression by functionally equivalent osteoblasts present at local areas of bone formation or of bone resorption.40 (E) QRT-PCR analysis of mRNA isolated from individual double-sorted Opn+CD45−Ter119− cells. Data are plotted as the percentage of osteoblasts (mean ± SD) with increased expression of OC, ALP, OA, RX2, or OS compared with a control population of single-sorted total BM cells (n = 2 independent experiments).

Phenotypic isolation of osteoblasts. (A) Strategy for isolation of bone-associated cells. (B) Flow cytometric analysis of single-cell suspensions from collagenase-treated bone (Bone) or BM aspirates (BM). Data are presented as dot plots showing staining for hematopoietic markers (CD45/Ter119) and Opn. The frequency of Opn+CD45−Ter119− cells is shown in the upper left. “-CNTRL” indicates representative background fluorescence seen in the absence of Opn secondary antibody (rat-αFITC) or when an isotype control (rat-αIgG) only is used. This is representative of 10 to 15 independent experiments. (C) QRT-PCR analysis of mRNA isolated from double-sorted Opn+ cells (OPN+) compared with irrelevant bone marrow cells (BM) or Opn− (OPN−) cells. Relative expression (mean ± SD) over a β-actin normalization control is shown for osteocalcin (OC), alkaline phosphatase (ALP), osteoactivin (OA), Runx2 (RX2), and osterix (OS), vascular endothelial growth factor receptor 1 and 2 (VEGFR1, VEGFR2), tyrosine kinase with Ig and EGF homology domains 1 and 2 (TIE1, TIE2), endothelial nitric oxide synthase (eNOS); n = 3 independent experiments for osteolineage markers; n = 2 independent experiments for endothelial markers). Each transcript was measured in triplicate. (D) Flow cytometric analysis of parathyroid hormone receptor-1 (PTHR-1) expression by Opn+CD45−Ter119− cells (gray histogram) or BM cells (black line). Both PTHR1hi and PTHR1lo OPN−CD45−Ter119− cells exhibited equal levels of activity of bone-specific ALP (Figure S2), consistent with an equivalent state of osteolineage differentiation of PTHR1hi and PTHR1lo cells12,42  and probably reflecting known differences in PTHR1 expression by functionally equivalent osteoblasts present at local areas of bone formation or of bone resorption.40  (E) QRT-PCR analysis of mRNA isolated from individual double-sorted Opn+CD45−Ter119− cells. Data are plotted as the percentage of osteoblasts (mean ± SD) with increased expression of OC, ALP, OA, RX2, or OS compared with a control population of single-sorted total BM cells (n = 2 independent experiments).

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